Formation of isoprene in plants is due to the presence of an isoprene synthase (IspS) gene (Miller et al., Planta 213: 483-487, 2001), a nuclear gene encoding for a chloroplast-localized protein that catalyzes the conversion of one of the products of the MEP pathway, dimethylallyl diphosphate (DMAPP), to isoprene (Lichtenthaler, Biochem Soc Trans 28: 785-789, 2000). Plant isoprene synthases, encoded by the IspS gene, have been cloned and characterized from poplar (Populus alba; Populus tremuloides) (Miller et al. 2001, supra; Sasaki et al., FEBS Lett 579: 2514-2518, 2005; Sharkey et al., Plant Physiol 137: 700-712, 2005) and kudzu vine (Pueraria montana) (Sharkey et al., 2005). Isoprene is a small hydrophobic and volatile molecule that can easily go through cellular membranes and thereby be emitted from the leaves into the atmosphere. The process of heat stress-induction and emission of such short-chain volatile hydrocarbons by plants has been discussed as undesirable pollution of the atmosphere in the literature (Sharkey et al., Ann Bot (Lond) 101: 5-18, 2008). It has been shown that isoprene production and release can function as a protective mechanism for the plant via which to increase thermo-tolerance (Sasaki et al., Plant Cell Physiol 48: 1254-1262, 2007; Sharkey et al., Plant Physiol 125: 2001-2006, 2001; Singsaas et al., Plant Physiol 115: 1413-1420, 1997). It has also been described that volatile hydrocarbons can be produced using microalgae, cyanobacteria, or bacteria that have been engineered to express IspS genes (WO 2008/003078).
Cyanobacteria express the MEP pathway. The corresponding enzymes are involved in the biosynthesis of a variety of molecules (e.g., carotenoids, tocopherols, phytol, sterols, hormones, among many others). However, unlike certain herbaceous, deciduous and conifer plants, cyanobacteria do not have isoprene synthase.
The MEP isoprenoid biosynthetic pathway in bacteria typically uses pyruvate and glyceraldehyde-3-phosphate as substrates, which are combined to form deoxyxylulose-5-phosphate (DXP). DXP is then converted into methyl-erythitol phosphate (MEP), which is subsequently modified to form hydroxy-2-methyl-2-butenyl-4-diphosphate (HMBPP). HMBPP is the substrate required for the formation of IPP and DMAPP as the final step in this biosynthetic pathway (FIG. 1). Cyanobacteria also contain an IPP isomerase that catalyzes the inter-conversion of IPP and DMAPP (Barkley et al., J Bacteriol 186: 8156-8158, 2004; Poliquin et al., J Bacteriol 186: 4685-4693, 2004). Genetic inactivation of the IPP isomerase gene results in impairment of isoprenoid biosynthesis from photosynthetic substrates (Poliquin et al., 2004, supra).
Previous studies with Synechocystis sp. PCC 6803 have shown that, under photosynthetic growth conditions, substrate for the MEP pathway in cyanobacteria may not derive from pyruvate and G3P. Rather, substrates originating from the pentose phosphate cycle may enter the pathway at steps later than MEP (Ershov et al, J Bacteriol 184: 5045-5051, 2002; Poliquin et al. 2004, supra) (FIG. 1), providing a more direct link between products photosynthesis and the isoprenoid biosynthetic pathway. Furthermore, it has been shown that, in the cyanobacterium Thermosynechococcus elongatus BP-1, the reaction catalyzed by GcpE, an Fe—S containing enzyme responsible for the formation of HMBPP, is dependent upon reduction by ferredoxin for its activity (Okada and Hase, J Biol Chem 280: 20672-20679, 2005) (FIG. 1), providing yet another direct link between photosynthesis and the isoprenoid biosynthesis pathway.
This invention in based, in part, on the discovery of nucleic acids and expression systems that provide production of short-chain hydrocarbons using genetically engineered cyanobacteria, e.g., Synechocystis sp. PCC6803, to generate improved new strains capable of isoprene (C5H8) production. Such genetically modified cyanobacteria can be used commercially in an enclosed mass culture system, e.g., (photo)bioreactors, to provide a source of renewable fuel for internal combustion engines or, upon on-board reformation, in fuel-cell operated engines; or to provide a source of isoprene for use in other chemical processes such as chemical synthesis.